1. Field of the Invention
The present invention relates to an induced pluripotent stem cell (iPSC) model of Fabry disease, a preparation method thereof, and a use of the same for the study of Fabry disease development and for the screening of a therapeutic agent for the disease.
2. Description of the Related Art
Fabry disease is a X-gene related recessive disorder caused by mutation of GLA that is the gene encoding α-galactosidase (GLA). The said GLA gene resides on xq22.1 of #7 exon, and encodes a glycoprotein that is composed of 370 amino acids which is processed from a precursor protein composed of 429 amino acids (Schiffmann, R. Pharmacology & therapeutics 122, 65-77 (2009)). According to the previous report, the incidence rate of Fabry disease is 1 out of 117,000 males (Meikle, P. J. et al. Jama 281, 249-254 (1999)). However, according to the most recent screening, the incidence rate is rapidly increasing and is diagnosed in 1 boy out of 3100˜3700 boys (Spada, M. et al. American journal of human genetics 79, 31-40 (2006)).
Once the lysosomal enzyme is deficient because of GLA gene mutation, globotriaosylceramide (Gb3, CD77) which is the neutral glycosphingolipid known to be acting as a Shiga toxin receptor in Burkitt's lymphoma cells is excessively accumulated, which seems to be the reason of Fabry disease (Nudelman, E. et al. Science New York, N.Y 220, 509-511 (1983)).
Up to date, approximately at least 400 mutation sites have been known as the possible mutation site in GLA gene (http://www.hgmd.cf.ac.uk). Symptoms of Fabry disease vary from the mutation site of GLA gene. In most mutation cases, α-galactosidase activity disappears. Missense mutations take 5˜10%, and clinically important major pathophysiology is not caused with the remaining enzyme activity (Clarke, J. T. Annals of internal medicine 146, 425-433 (2007)).
The major pathophysiological symptom shown in Fabry disease patients is Gb3 accumulation in various cells such as vascular cells, cardiac cells, kidney epithelial cells, and neuronal cells. In particular, Gb3 accumulation in vascular cells causes systemic cardiovascular dysfunction exemplified by stroke or myocardial infraction.
The only treatment method for Fabry disease is repetitive enzyme replacement therapy, wherein the α-galactosidase ‘Fabrazyme’ belonging to agalsidase beta (Eng, C. M. et al. The New England journal of medicine 345, 9-16 (2001)) or ‘Replagal’ belonging to agalsidase alpha (Schiffmann, R. et al. Proceedings of the National Academy of Sciences of the United States of America 97, 365-370 (2000)) is administered to Fabry disease patient to eliminate Gb3 accumulated in various cells. The intravenously injected enzyme is introduced in cells via mannose 6 phosphate (M6P) receptor in plasma membrane, and then further moves to lysosome. The administration of such therapeutic enzyme plays a key role in treating Fabry disease. However, those recombinant enzymes like Fabrazyme and Replagal are unstable in blood and might cause allergic reaction if they are repeatedly administered (Eng, C. M. et al. The New England journal of medicine 345, 9-16 (2001); Schiffmann, R. et al. Proceedings of the National Academy of Sciences of the United States of America 97, 365-370 (2000); Sakuraba, H. et al. Journal of human genetics 51, 180-188 (2006)).
To study Fabry disease and to develop a treating agent for the disease, it is the general manner to establish GLA knock-out mouse and to investigate the involvement of Gb3 accumulation in endothelial dysfunction. The abnormal accumulation of Gb3 was observed in caveolae of aortic endothelial cells in the GLA knock-out mouse (Shu, L. & Shayman, J. A. The Journal of biological chemistry 282, 20960-20967 (2007)). According to the previous report, such abnormal Gb3 deposit induced dysfunction of calcium channel in the GLA knock-out mouse endothelial cells (Park, S. et al. Cardiovascular research 89, 290-299 (2010)).
Even though vasculopathy was found in the GLA knock-out mouse as the Fabry disease model system caused by the lack of α-galactosidase A from various research attempts, the precise mechanism of cardiovascular complication induced by the deficiency of α-galactosidase A activity and the resulting Gb3 deposit in vascular cells has not been disclosed, yet. It is expected that the limit of study on the outbreak mechanism of Fabry disease can be overcome by using iPSCs originated from Fabry disease patient somatic cells.
Stem cells are the cells in the phase of pre-differentiation before being differentiated into each tissue forming cells, which can be obtained from the tissues of an embryo, a fetus, and an adult. Stem cells have self-proliferative activity that makes unlimited proliferation possible from undifferentiated status and have pluripotency, so that they can be differentiated into various tissue cells once a certain stimulus is given. That is, stem cells become differentiated by a certain differentiation stimulus (environment), and are self-renewal so as to produce those cells that are same as themselves by cell division, unlike the differentiated cells whose cell division has been finished. Stem cells also have plasticity, by which stem cells can be differentiated into different cells when the environment is changed or when a different stimulus is provided.
Human pluripotent stem cells including induced pluripotent stem cells (induced pluripotent stem cells; iPSCs) are characterized by pluripotency which is the ability to be differentiated into various types of cells. Therefore, when iPSCs are used for in vitro differentiation system, they can be efficiently used for the evaluation of not only therapeutic potential owing to the low risk of immune rejection but also complicated disease mechanism particularly in relation to the early development stage of organogenesis (Muotri, A. R. (2009) Epilepsy Behav 14 Suppl 1: 81-85; Marchetto, M. C., B. Winner, et al. (2010) Hum Mol Genet 19(R1): R71-76).
It has been reported that when the iPSCs originated from patients having different genetic diseases were induced to be differentiated into disease-related cells, the cells displayed disease-specific phenotype (Park, I. H. et al. Cell 134, 877-886 (2008); Tiscornia, G. et al. Nature medicine 17, 1570-1576 (2011)). That is, such disease-specific iPSCs can be differentiated into disease-related cells and therefore they can be effectively used for the study of specific mechanism of disease and for the screening of a therapeutic agent.
Thus, the present inventors tried to establish a stem cell model for the study of Fabry disease. In the course, the inventors induced the development and differentiation of Fabry disease originated induced pluripotent stem cells (iPSCs), embryoid body (EB), and vascular cells from the fibroblasts of Fabry disease patient and confirmed that Gb3 was accumulated in the iPSCs by the significantly reduced expression and activation of GLA protein. The inventors also induced the differentiation of vascular cells from Fabry disease originated iPSCs. As a result, the said iPSCs were differentiated into vascular endothelial cells and vascular smooth muscle cells wherein the marker protein was significantly expressed. When the vascular endothelial cells and vascular smooth muscle cells were treated with Fabrazyme, the accumulation of globotriaosylceramide (Gb3, CD77) was significantly reduced. Therefore, the present inventors confirmed that the Fabry disease cell modeling method using induced pluripotent stem cells (iPSCs) of the invention can be efficiently used for the study of Fabry disease development and for the screening of therapeutic agent candidates, leading to the completion of this invention.